The present invention relates generally to the treatment of viral infections using lipid derivatives of antiviral compounds. More particularly, the present invention relates to lipid derivatives of antiviral phosphonoacids and their use. The lipid derived compounds can be integrated into the structure of liposomes, thereby forming a more stable liposomal complex which can deliver greater amounts of these compounds to target cells with less toxicity.
The publications and other reference materials referred to herein are hereby incorporated by reference, and are listed for convenience in the bibliography appended at the end of this specification.
There has been a great deal of interest in recent years in developing agents to treat viral infections. In the past the most significant viral diseases were those caused by viruses of the herpes and influenza groups of viruses as well as those viruses causing hepatitis. In the past decade, infections with human immunodeficiency retrovirus (HIV) have become a major public health problem. Effective antiviral agents are those that interfere with the replication or transcription of viral genetic information while not inhibiting the normal functions of the host cell.
Phosphonoacetic acid (PAA) and phosphonoformic acid (PFA or Foscarnet), having the following structures: ##STR1## have been shown to have good antiviral activity against herpes simplex viruses types 1 and 2 (1), as well as against influenza viruses, hepatitis virus B, and retrovirus infections (2).
Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). HIV is a retrovirus which infects cells bearing the CD4 (T4) surface antigen, such as CD4+ helper lymphocytes, CD4+ monocytes and macrophages and certain other CD4+ cell types. The HIV infection of CD4+ lymphocytes results in cytolysis and cell death which contributes to the immunodeficiency of AIDS; however, CD4+ monocytes and macrophages may not be greatly harmed by the virus. Viral replication in these cells appears to be more prolonged and less cytotoxic than in lymphocytes, and as a result, monocytes and macrophages represent important reservoirs of HIV infection. It has recently been discovered that macrophages may serve as reservoirs of HIV infection even in certain AIDS patients who test negative for the presence of HIV antibodies. No effective cure is available for AIDS, although nucleosides analogues, particularly the dideoxynucleosides have been shown to prolong life and to reduce the incidence of certain fatal infections associated with AIDS.
Dideoxynucleoside analogues are the most potent agents currently known for treating AIDS, but these therapies are not entirely satisfactory. In a recent human clinical trial using AZT, serious toxicity was noted, evidenced by anemia (24%) and granulocytopenia (16%) (3,4). Certain monocyte-derived macrophages, when infected with some strains of HIV, have been found to be resistant to treatment with dideoxycytidine, azidothymidine, and other dideoxynucleosides in vitro as shown by Richman, et al. (5). The resistance may be due in part to the low levels of dideoxynucleoside kinase which result in a reduced ability to phosphorylate AZT, ddC or ddA.
Phosphonoformate (PFA) may provide an effective alternative therapy to nucleoside analogues. PFA inhibits a broad range of DNA polymerases as well as the RNA polymerase of influenza virus. PFA also inhibits the reverse transcriptase (RT) of HIV and other retroviruses at concentrations below 1 .mu.M. Since the DNA polymerases are much less sensitive to PFA than the reverse transcriptases, the possibility exists that this drug may have a good therapeutic ratio for use in HIV infection. The phosphonoacids PFA and PAA may also supplement therapy using antiviral nucleosides. Lambert (6) has found that when PFA or PAA are coupled with certain antiviral nucleosides, particularly 5-bromo-2'-deoxyuridine (BUdR), the antiviral activity of the coupled nucleoside against herpes simplex viruses is greater than that of the parent nucleoside.
Efforts to increase the effectiveness of both antiviral nucleosides analogs and the phosphonoacids include association of these agents with lipids.
Attempts have been made to improve the therapeutic effectiveness of nucleoside analogues, such as arabinofuranosylcytosine (ara-C) and arabinofuranosyladenine (ara-A) as chemotherapeutic agents in the treatment of various types of cancer, by chemically linking them to phospholipids in order to enhance their catabolic stability (7). These phospholipid-derived agents showed a decreased toxicity and increased stability over the nucleoside analogues alone. However, they also exhibited poor cellular uptake (6) and poor drug absorption (8).
Another approach to increase the effectiveness of antiviral agents comprises encapsulation within liposomes to facilitate their delivery to cells. Liposomes are lipid vesicles which can be formed according to the method of Alex Bangham. Bangham and coworkers discovered in 1965 that dried films of phosphatidylcholine spontaneously formed closed bimolecular leaflet vesicles upon hydration (9). One of the most effective applications of liposomes in medicine is as a carrier to deliver therapeutic agents to target organs. The agents are encapsulated during the process of liposome formation and released in vivo after liposomes are taken up by cells. Liposomes thereby provide a means of delivering higher concentrations of therapeutic agents to target organs. Further, since liposomal delivery focuses therapy at the site of liposome uptake, it reduces toxic side effects.
Liposomal incorporation has been shown to provide a more effective way of delivering antiparasitic compounds which not only increases the potency of the dose but prolongs its efficacy and decreases its toxicity. For example, liposomal antimonial drugs are several hundred-fold more effective than the free drug in treating leishmaniasis as shown independently by Black and Watson (10) and Alving, et al. (11). Liposome-entrapped amphotericin B appears to be more effective than the free drug in treating immunosuppressed patients with systemic fungal disease (12). Other uses for liposome encapsulation include restriction of doxorubicin toxicity (13) and diminution of aminoglycoside toxicity (14).
PFA has been found to inhibit HIV-1 replication in several in vitro systems at concentrations which are attainable in patients. However, the low degree to which PFA enters cells causes much higher levels to be required than that found to be effective in cell free systems with HIV RT (15). Also, PFA has toxic side effects and is known to accumulate in bone because of its similarity to pyrophosphate. Phosphonoacetic acid (PAA), which has antiviral activity similar to that of PFA, appears to have an affinity for bone that may preclude its use in humans (6).
Attempts have been made to increase the intracellular antiviral efficacy of the phosphonoacids by encapsulating them into liposomes. Szoka, F. and Chu, C. (16) found that liposomal delivery enhanced the cellular uptake and viral inhibitory activity of both PFA and PAA. Liposomal encapsulation also decreased the cytopathic effect of PFA; however, the cytopathic effect of liposomal PAA as compared to the free drug appeared to be increased.
As previously mentioned, it is now thought that macrophages are an important reservoir of HIV infection (17, 18). Macrophages are also a primary site of liposome uptake (19, 20). Accordingly, it would be desirable to utilize liposomes to enhance the effectiveness of antiviral phosphonoacid and antiviral phosphonoacid esters of antiviral nucleosides in treating AIDS and other viral infections. Clearly, it would be useful to have more effective ways of delivering large amounts of effective antiviral phosphonoformate compounds to macrophages infected with HIV or other viruses and to other cells having viral infections.
Co-pending applications, U.S. Ser. Nos. 216,412 and 319,485 (21, 22) disclose lipid derivatives of nucleoside analogues which are capable of being incorporated into the structure of liposomes so as to further improve therapy comprising liposomal delivery of these agents.
In order to use phosphonoacid antivirals more effectively, it is desirable to synthesize lipid prodrugs of the agents. It is therefore an object of the invention to provide methods for producing phosphonoacid lipid derivatives which can be incorporated into stable liposomal form.
The methods disclosed here apply not only to the use of lipid derivatives of phosphonoacids in the treatment of AIDS and other retroviral diseases, but also to their use in the treatment of diseases caused by other viruses, such as influenza, herpes simplex virus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis B virus, Epstein-Barr virus (EBV), and varicella zoster virus (VZV).